Counter-attack of biocontrol agents: Environmentally benign Approaches against Root-knot nematodes (Meloidogyne spp.) on Agricultural crops

Root-knot nematodes (Meloidogyne spp.) are obligate sedentary endoparasites, considered severe crop-damaging taxa among all plant-parasitic nematodes globally. Their attacks through parasitic proteins alter the physiology and machinery of the host cells to favour parasitism and reduction in crop yield. Currently, the use of excessive pesticides as a fast remedy to manage this pest is hazardous for both the environment and humans. Keeping this view in mind, there is an urgent need for developing efficient eco-friendly strategies. Bio-control as an eco-friendly is considered the best approach to manage nematodes without disturbing non-target microbes. In bio-control, living agents such as fungi and bacteria are the natural enemies of nematodes and the best substitute for pesticides. Fungi, including nematode-trapping fungi, can sense host signals and produce special trapping devices viz., constricting rings and adhesive knobs/loops, to capture nematodes and kill them. Whereas, endo-parasitic fungi kill nematodes by enzymatic secretions and spore adhesion through their hyphae. Bacteria can also control nematodes by producing antibiotic compounds, competing for nutrients and rhizosphere, production of hydrolytic enzymes viz., chitinases, proteases, lipases, and induction of systemic resistance (ISR) in host plants. Scientists throughout the world are trying to evolve environmentally benign methods that sustain agricultural production and keep nematodes below a threshold level. Whatever methods evolve, in the future the focus should be on important aspects like green approaches for managing nematodes without disturbing human health and the environment.


Introduction
The rhizosphere is the residence of several microbes that interact with each other and the plant roots.Some of them are advantageous for plants and soil microbes while others are deleterious.Such deleterious microbes are considered pests that incite biotic stress and lower the productivity of affected crops [1].Plant-parasitic nematodes (PPNs) are one of the most serious plant-damaging soil microbes for agricultural production worldwide [2,3].Currently, around 4100 species of PPNs have been discovered that affect various crops like fruits including grapevine, bananas, sugarcane, and vegetables viz., brinjal, chili, tomatoes, cauliflower, cabbage, carrot, okra, maize, cotton, potatoes and soybeans [4,5].The impact of PPNs is more observed in vegetable-producing countries viz., Bangladesh, India, Pakistan, and some areas of African countries where the major issues are poverty, hunger, and inadequate food supply.According to nematologists, the top 10 PPNs can significantly damaging global agriculture production; these are root-knot nematodes (Meloidogyne spp.), root lesion nematode (Pratylenchus spp.), cyst nematodes (Heterodera and Globodera), pine wilt nematode (Bursaphelenchus xylophilus), burrowing nematode (Radopholus similis), rice white tip nematode (Aphelenchoides besseyi), reniform nematode (Rotylenchulus reniformis), stem and bulb nematode (Ditylenchus dipsaci), dagger nematode (Xiphinema index-the virus vector nematode) and false root-knot nematode (Nacobbus aberrans) [6].However, root-knot nematodes (Meloidogyne spp.) are the most important and dangerous soil-borne pathogen among all PPNs (Khan et al., 2023).

Biotic stress
Biotic stress is the response of phytopathogenic infection, especially root-knot nematodes (RKNs), fungi, and bacteria that bring down productivity and crop yield [7].Such harmful pests viz., RKNs, acquire their nutrition from the host plant and cause the weakening and killing of host cells [8].Plants' reply to such biotic stress is complicated, involving interaction between two living organisms.Simultaneously, plants evolve protective and defensive mechanisms against such pathogenic interactions.Thus, pathogen-plant interaction is a multifaceted method where actual interaction takes place between plant and pathogen-derived stimuli viz., proteins, genes, sugars and lipopolysaccharides.Such stimuli obtain from pathogens are responsible for the severity of infection and pathogenicity which is determined by the pathogen's ability to colonize the plant cell [9].

Root-knot nematodes (Meloidogyne spp.) as a major biotic stress-causing agent
RKNs (Meloidogyne spp.) are major biotic stress-causing agents for several crops globally.It belongs to the order of Tylenchida, ubiquitous in nature and sedentary endoparasites that depend on the host for completing their life cycle [10].More than 100 species of RKNs have been discovered that affect almost 3000 plants, including vegetables and fruits, globally [11].Among them, four species of RKNs viz., M. javanica, M. incognita, M. hapla and M. arenaria are major and harmful, causing more damage up to 90 % to the plants and making them more susceptible to other pathogens [12,13].Fourteen root-knot species have been recognized from distinct places in India [14].Out of which, M. incognita is one of the more damaging species among all, causing major loss and reduction in vegetables yield and quality viz., tomato, okra, eggplant, cauliflower, cabbage and spinach [15].However, it has been also observed that certain fields in India were predominantly occupied by root-knot disease due to which vegetable crops were affected severely (Khan et al., 2022).Other species viz., M. graminicola, M. javanica, and M. arenaria are also pathogenic and common [14].However, M. hapla is restricted to temperate/cooler areas [16] (Table 1).

Biology of root-knot nematodes: the journey from soil to host
RKNs exhibited sexual dimorphisms, i.e., the females are pear-shaped and the males are free-living while second-stage juveniles (J2s) are infective.The life cycle of the RKNs is completed in 25-28 days at 27 • C temperature and divided into different stages (viz., eggs, juveniles, and adults) but this period is changed due to soil moisture (lesser extent), soil temperature (widely) and availability of a suitable host [11] (Fig. 1).The successful completion of the life cycle, involving sequential molts from egg to adult, includes morphologically and functionally distinct stages.In optimal conditions, a single female can produce 200-500 eggs in a mucilaginous matrix [11].Embryogenesis converts eggs into J1s, which remain inside the eggshell.Following the first molt, J1 transforms into J2s, which comes out in the soil from the eggshell to search for a host plant [29].By using chemosensory amphids, J2s migrate toward the susceptible host roots by sensing chemical gradients secreted by the roots [30].During the compatible interaction with the susceptible plants, J2s penetrate the roots and migrate straight down, between the cortical cells, to the apical meristematic site.Then they start moving upwards in the vascular bundle, inducing specialized hypertrophied feeding cells known as giant cells (GCs).These GCs undergo multiple mitotic cycles without cytokinesis, which causes them to enlarge and become multinucleated [31].GCs are the places, where nematodes complete their life cycle and occupy sedentary habitats [32].They possess a hollow, protruding stylet at their anterior end, which they utilize to inject secretions into and extract nutrients from GCs.The hyperplasia of the root cells leads to irregular growth and causes the formation of galls or root-knot, which is a unique feature of root-knot disease caused by RKNs [33] (Fig. 2).

Effector proteins of root-knot nematodes: favour parasitism by altering the physiology and metabolism of the host cells
Effector proteins of RKNs are pathogenic in nature that parasitize the host plant by changing the metabolism and physiology of the host cell.Parasitism and manipulation of host cellular mechanisms are favoured by the interactions of effector proteins with certain host proteins.Such interactions viz., parasitic proteins-host proteins also suppress and weaken the defence and defence-related genes of the host [34].During parasitism, J2s release oesophageal parasitic proteins through their stylet (piercing organ) into the host tissues and induce GCs formation [35].However, J2s release such parasitic proteins either on the cell surface or directly within the cytoplasm of the host cell.Both of the cases mimic the few proteins that result in the changes in the expression of host cell genes [36].Effectors proteins, cell wall digesting enzymes (CWDEs), and the proteins that mimic the proteins of the host are important secretions of RKNs for the successful feeding and their establishment on the host [37,38] (Table 2).

Modes of action of effector proteins
The cell wall of the host plant acts as the first barrier for the entry of any pathogen, including RKNs.For the successful establishment and crossing of such a barrier, RKNs release several CWDEs for the damaging cellular constituents of the host cell.In addition to    CWDEs, RKNs release a few other enzymes to favour parasitism within the host viz., polygalacturonase, pectate lyases, 1, 4 β endoglucanase, endo 1,4 β xylanase, expansins, and a cellulose binding protein [35,[54][55][56][57].Some of the effector proteins are released by J2s through their three oesophageal glands including two sub ventral glands [58].The recipient of the secretory substances of RKNs is located in the apoplasm of the host plant [43].

Inhibition of the defence-related plant hormones of the host
Plant hormones such as auxin (indole acetic acid, IAA), ethylene, jasmonic acid (JA), cytokinin and salicylic acid (SA) play an important role in the growth and development of plants.Several functions like cell differentiation, elongation, cell expansion and response to various biotic and abiotic stresses are mediated by ethylene and auxin [59].Such hormones play an impressive role in phytopathogenic interactions.An effector, Mi-CM is released by M. incognita that changed the auxin pool of the host plant [60].Recently, a few researchers reported that M. incognita possess an effector protein MiISE6 engaged in the plant-nematode interaction.This effector protein plays a major role during the initial stages of parasitism by M. incognita and obstructs several signalling pathways viz., JA pathways of the host plant [41].Another parasitic protein Mi-CM-3 was also found in M. incognita which inhibits the SA pathway and plant immune system of the host plant and favours parasitism within the host [48].

Suppression of effector-triggered immunity and/or pathogen-triggered immunity of the host
Few effector proteins viz., MiMsp40 from M. incognita suppress the immune system viz., effector-triggered immunity (ETI) and pathogen-triggered immunity (PTI) of the host plant and enhance nematode parasitism [50].Mi-CRT (calreticulin) is another effector released by nematodes into the apoplasm of the host tissue that causes the inhibition of the PTI triggered by the PAMP elf8 in Arabidopsis thaliana [51].

Mimicry and manipulation of the defence-related proteins of the host
Meloidogyne graminicola is a very harmful nematode for rice growth and brings down rice production.An important effector protein, Mg16820 obtained from M. graminicola, reacts as a suppressor of the immune system of the host cell and shows an effective role in nematode parasitism.Furthermore, Mg16820 inhibits flg22-induced reactive oxygen species (ROS) in the rice plant that participate in the stress response and also suppress the R2/Avr2-and Mi-1.2-induced hypersensitive response [52].One more effector named, MiPFN3 (M.incognita Profilin 3) was obtained from J2s of M. incognita.MiPFN3 is inoculated within the host plant through the stylet of M. incognita where it favours and stimulates nematode parasitism [53].MiIDL1 (M.incognita IDAlike 1 effector) a potent effector protein of M. incognita that mimic with functional IDA (Inflorescence deficient in abscission) or IDA like genes of Arabidopsis for favouring parasitism in Arabidopsis plant in the form of root galls [37].Lin et al. [47], found an effector MjTTL5 from M. javanica that speeds up parasitism by attacking the ferredoxin/thioredoxin system of the host.

Changes in the host cell metabolism
GCs formation in host roots by RKNs influence the metabolic mechanisms of the host plant because such cells act as feeder cells and source of nutrients and metabolites for developing nematodes.Few cells in the vicinity of GCs also become large due to the accumulation of huge amounts of nutrients.Such neighbouring cells provide a direct approach for food to RKNs.Eloh et al. [61] observed the changes in metabolic pathways when plants are infected with RKNs.The latter's infection also changes the methods of protein formation in host roots [62].
By examining these all changes in the host's cell physiology and metabolism, including translation it has been necessary to develop effective eco-friendly management strategies against RKNs.

Bio-control methods against root-knot nematodes parasitism
The intensity for selecting microbes as bio-control agents (BCAs) has grown day by day due to their pest-managing proprieties becoming a key factor in integrated nematode management (INM) on crops [63].The rhizosphere harbours numerous microbes which provide resistance to plants against several pathogens, including RKNs [64].However, the bio-control of RKNs has been researched for more than a decade.It might be recognized as a multi-trait wonder whose achievement relies upon the rhizospheric competition and interactions with other microbes, the adjustment in different environmental situations and more defence of the plant against several pathogens including RKNs [65,66].Uses of chemical-based pesticides are the fastest methods to control plant disease however it is very noxious to the environment, human being and soil also decrease soil fertility and make soil disintegrate.So, there is a need to find out substitutes for pesticides to control plant diseases caused by PPNs.Bio-control is considered as a good alternative for chemical-based nematicides and manages plant disease in an eco-friendly manner and also increases soil fertility without disturbing fauna and flora [67,68].Among bio agents, fungi (arbuscular mycorrhizal, nematophagous fungi, and Trichoderma spp.) and bacteria are considered potent bioagents against RKNs and other PPNs.Besides whole microbes sometimes derivatives viz., microbial metabolites and their products can also utilize as BCAs [69,70].

Fungi-mediated bio-control against root-knot nematodes
Fungi and their metabolites are found to be control the population of PPNs, which are detrimental to plant health.Trichoderma spp.well-known individuals recognized as important BCAs against RKNs, parasitize the infective juveniles and inhibit root penetration by nematodes also enhance the growth and yield of crops [71].To prevent the crop from nematode infestation Trichoderma spp.must be A. Khan et al. applied in the soil before crop planting because it completely colonizes the roots [72].However, the addition of organic matter to soil (e.g., chicken litter) can enhance the bio-control activity of Trichoderma [73].Other groups' viz., nematophagous and arbuscular fungi also possess nematicidal activity against RKNs.The bio-control activity of BCAs depends on the nematode species, host plant, its exudates, and other rotation crops [74].

Nematophagous fungi as bio-control agents and their modes of action
Fungi that feed on PPNs are considered as nematophagous fungi.These fungi are classified into four groups based on the mechanisms of action against nematodes: (i) predatory (nematode trapping) fungi make extensive constricting rings and hyphal networks as trapping tools for capturing nematodes, eg., Arthrobotrys oligospora and Drechslerella sp., (ii) endoparasitic fungi (obligate parasites of nematodes) parasitize nematodes either by direct ingestion or attaching to their surface followed by growth, germination, and killing of nematodes, eg., Drechmeria coniospora, (iii) nematode's eggs and female parasitic fungi, which as facultative parasites of sedentary stages of nematodes such as cysts and eggs, eg.Pochonia chlamydosporia (Metacordyceps chlamydosporia), P. rubescens and Paecilomyces lilacinus and (iv) toxins producing fungi inhibit the nematodes mobility before hyphae penetrate the nematode cuticle, eg.Pleurotus ostreatus [75,76].However, few nematophagous fungi exist as facultative saprotrophs, i.e., taking their nutrition from dead and decaying organic matter in the absence of nematodes, and therefore organic matter rich soil boost their existence [75].

Arthrobotrys oligospora
A. oligospora is an important and most common nematophagous fungus.This fungus traps nematode juveniles facultatively for its nitrogen requirement.It also obtains carbon and energy-rich foods from dead and decomposed organic matter [77].In vitro experiment shows the high efficiency of A. oligospora in the killing and capturing of M. incognita J2s.The microscopic study also revealed that the J2s of RKNs were trapped by adhesive loops of A. oligospora [78].Moreover, A. oligospora treatment significantly reduces the disease development caused by RKNs on tomato plants and shows 74 % predation against M. incognita in comparison to 36 % of the control (having only sterilized distilled water without A. oligospora) [79].Fig. 3. Modes of action of nematophagous fungi as bio-control agents against root-knot nematodes on agricultural crops.
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Drechmeria coniospora
Fungus D. coniospora infects nematodes with their spores (zoospores or conidia) via the attachment of conidia to the nematode cuticle and inserting the hyphae in nematode epidermis and killing them [80].However, conidia attach to various PPNs such as Cephalenchus sp., Pratylenchus penetrans, Heterodera schachtii, and Ditylenchus spp.but the attachment of spores to any nematode species does not favour its killing and infection [76,80].

Pochonia chlamydosporia, Paecilomyces lilacinus and Lecanicillium psalliotae
P. chlamydosporia, P. lilacinus, and L. psalliotae are considered as potent BCAs among all female and egg parasitic fungi of RKNs and cyst nematode.These fungi mainly attack nematode females and their eggs because these are the main targets.However, J2s of RKNs inside egg masses can also be attacked by P. chlamydosporia [81,82].A few strains of P. chlamydosporia also stimulate the defence in tomato plants by activation of the salicylic acid (SA) pathway against M. incognita.It has been observed that co-inoculation of both fungus and nematode in tomato plants revealed the expression of defence-related protein 1 (PR1), lipoxygenase (LoxD) genes, pathways viz., jasmonic acid (JA) and SA pathways which provide resistance against M. incognita.In this co-inoculation experiment, fungal chlamydospores were inoculated in the soil just one week before the inoculation of M. incognita J2s [83].P. lilacinus is another nematode egg parasitic fungus, used as BCAs against RKNs [28].According to Kiewnick and Sikora [84], inoculation of P. lilacinus strain 251 (PL251) in tomato field infested with M. incognita reduces the egg masses by 74 %, root galling by 66 % and the population of nematodes by 71 % compared to untreated control.However, P. lilacinus directly infect eggs and sedentary stages of nematodes by producing few nematicidal proteins viz., chitinases, leucinotoxins, acetic acid, and proteases [85].

Pleurotus ostreatus
P. ostreatus (oyster mushroom) as a toxin-producing fungus having the ability to kill nematodes by paralyzing them.This fungus feeds on nematodes for their N 2 requirement under low nutrient conditions.However, current studies show that P. ostreatus kill nematodes by increasing calcium (Ca) influx and necrosis in the neuromuscular system of Caenorhabditis elegans through nematode sensory cilia [86,87].A few studies also revealed the bio-control efficiency of P. ostreatus against PPNs including root-knot nematode, M. incognita and sugar beet nematode, Heterodera schachtii in cowpea [88,89] (Fig. 3).

Trichoderma spp.
Trichoderma spp.classified as cosmopolitan, saprotrophic, and widely applicable fungus.Consider as dominant among all known bio-control fungi against RKNs, and colonize the plant-soil ecosystems.They have ability to protect plants from RKNs and other soilborne pathogens by colonizing the rhizosphere [90].They grow easily and are propagated on synthetic media under lab conditions, improving plant growth and inducing resistance against RKNs [91].They enhance the nutrient uptake from the soil in the inoculated plant [92].Trichoderma spp.also prevents nematode penetration by parasitizing the nematode egg shell and cuticle through its conidia and improves plant growth as well [93].The attachment affinities to Meloidogyne spp.eggs, cuticle, or gelatinous matrix of egg masses are species-specific [94].The most effective bio-control properties are mainly attributed to the different species viz., T. viride, T. citrinoviride (Snef1910 strain), T. virens, T. harzianum (strain MZ025966), T. koningii, T. longibrachiatum, T. asperellum, and T. polysporum which have a significant impact on disease development caused by RKNs.

Modes of action of Trichoderma spp.
In addition to competition for food and shelter with RKNs, Trichoderma spp.also shows antagonistic activity against harmful fungi [95] possessing a high ability to adapt to different environmental conditions and reducing the effect of harmful pathogens by their fast-growing rate [96,97].However, the current knowledge about the Trichoderma induced resistance in plants against other biotic pathogens viz., bacteria, fungi, and viruses are unknown [91].Although, previous researches find out that Trichoderma mediated resistance in plants may play an impressive role in reducing the infection caused by RKNs and other harmful pathogens [98,99].Root colonization by Trichoderma spp.changes the metabolism and physiology of the host plant, resulting in the production of several secondary metabolites (SMs) that function as defense responses against RKNs [100,101].

Trichoderma spp. alleviates root-knot nematodes-induced oxidative stress
Reactive oxygen species (ROS) are continuously formed in plants but RKN infection increases ROS production [101,102].Excessive production of ROS at infection sites harms the cell membranes by membrane lipid peroxidation [101,103].However, low levels of Malondialdehyde (MDA) and electrolyte leakage by 30.85 % and 38.89 % were found in tomato roots treated with T. herzianum and RKNs simultaneously, compared with RKNs infested roots [104].To normalize cellular functions, it is necessary to keep the ROS below the threshold level by promoting antioxidant production, and acting as a ROS scavenger [102,105].

Trichoderma spp. increases the concentrations of secondary metabolites
Secondary metabolism plays an important role in the production of antioxidants and SMs required for defence and to lower the high level of ROS under unfavourable conditions.Importantly, several SMs viz., phenols, flavonoids, lignin, and cellulose act as antioxidants to increase resistance and protect host plants from RKNs infection [100,106,107].Yan et al. [104], found an increased level of flavonoids, cellulose and lignin in T. herzianum treated tomato plants infested with RKNs compared to the untreated plants.Ahmad et al. [71] also finds that the combined application of T. harzianum with fly ash improved the SMs profile and defence-related genes in chili (Capsicum annum) against RKNs.
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Trichoderma enhances the enzymatic activity and transcript levels of genes involved in secondary metabolism
Trichoderma harzianum treated plants showed an increased level of enzymes that participated in secondary metabolisms to provide resistance against PPNs.Key enzymes such as cinnamyl alcohol dehydrogenase (CAD), glucose-6-phosphate dehydrogenase (G6PDH), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), shikimate dehydrogenase (SKDH), guaiacol peroxidase (G-POD), 4coumarate CoA ligase (4CL), and caffeic acid peroxidase (CA-POD) involved in secondary metabolisms in treated tomato plants compared to untreated control [104].Many studies also suggest the importance of a few genes viz., CAD, cinnamate-4-hydroxylase (C4H), and PAL in the regulation of the plant defence and phenylpropanoid pathway against RKNs [108,109].Pathogenesis-related proteins (PRPs), such as peroxidases, chitinases, and β-1,3-glucanase shows nematicidal and antimicrobial activity [99,110,111].In vitro observations suggest these enzymes damage the major parts of the nematode cuticle, and eggshell and ultimately lead to death [112].

Trichoderma spp. promotes the content of jasmonic acid (JA) and salicylic acid (SA)
To obtain good results and insight into the mechanisms of Trichoderma-induced resistance against RKNs, it is necessary to modify the SA and JA contents in the tomato roots by inoculating Trichoderma in the roots.However, a high level of both SA and JA was found in RKN-infected plant roots compared to non-infected plants (Table 3).

Advantages of Trichoderma spp. other than bio-control mechanisms
Besides BCAs, Trichoderma spp. as a part of soil microbes, play a key role in the decomposition, and decontamination/removal of harmful substances, i.e., xenobiotic from the soil ecosystem.Decontamination/removal of harmful substances is considered as 'bioremediation' involving the use of microbes to change the toxic compounds into non-toxic while decomposition leads to the release of beneficial nutrients available for plants growth [124,125].According to Weber et al. [126], T. viride uses nitrogenous compounds, viz., (trinitrotoluene, TNT explosive) at 100 and 50 ppm doses to fulfil their nitrogen requirement for normal growth and development of plants.Besides, the utilization of nitrogenous explosives as a nitrogen source, Trichoderma spp.also has the ability to degrade unwanted hydrocarbons from the aquatic ecosystem and bio-remediate them [127].Few other researches also reported the use of T. harzianum strain T22, for the biodegradation of diesel fuel, allowing it to be used as a source of carbon [128].It has been found that the combined application of chemical fertilizers and Trichoderma into the soil increases plant productivity, nutritional quality, and reproductive and vegetative development.The overall role and application of different species of Trichoderma make it as potent BCAs, and soil nutrient mobilizer, improving the yield and quality of the crops (Fig. 4).

Table 3
Modes of action of fungal species and their strains against root-knot nematodes on agricultural crops.

M. javanica Tomato
In vivo [115] Purpureocillium lilacinum Direct inhibition of nematode and their life cycle

M. chitwoodi Tomato
In vitro, in vivo [117] Scutellospora heterogama Root colonization and competition for space and nutrients with nematode

M. incognita Tomato
In vitro, in vivo [120] Combined application of P. chrysogenum and P. chlamydosporia Reduction in egg hatching, gall indices, and egg masses, increase juveniles' mortality

A. thaumasia and Tolypocladium cylindrosporum
Increase parasitism of nematodes and reduction in galls, egg masses

Tomato
In vitro, in vivo [122] P. lilacinum Inhibition of disease and life cycle of the nematode

Bacteria-mediated bio-control against root-knot nematodes
Bacteria are microscopic organisms that grow in diverse environmental conditions, ranging from radioactive waste, hot water springs, soil, and the deep biosphere of the earth's crust [129][130][131].They also exist as symbionts and parasites on several plants and animals.Some bacterial genera viz., Bacillus, Pseudomonas, Serratia, Pasteuria, Burkholderia, Achromobacter, Arthrobacter, and Rhizobium possess nematicidal action and are considered as potent BCAs of RKNs.Few commercial products have also been obtained from bacteria that possess nematicidal potential [74].Besides BCAs of nematodes, bacteria also enhance plant growth by increasing nutrient uptake from the soil.
The whole bio-control mechanisms depend on the competition for food and space with nematodes, the production of antibiotic compounds and hydrolytic enzymes against nematodes, and the induction of systemic resistance (ISR) in host plants.Among these, antibiosis is the most widely used mechanism involving the production of volatile organic compounds (VOCs), toxins, and some antibiotics against nematodes.

Antibiosis
It involves the production of toxic compounds by microorganisms harmful to other microbes.During the trophophase of the cell cycle, the development of bacterial cells is ideally higher however the metabolites and antibiotic compounds are formed during the idiophase when the nutrients become drained and the cell has expanded extensively [132].Such compounds are beneficial for plant growth and development.

Rhizospheric competition
Competition for food and space between pathogenic and non-pathogenic microorganisms in the rhizosphere is known as rhizospheric competition.Rhizospheric competence also promotes the population of beneficial microbes in the vicinity of plant roots.However, these beneficial soil microbes protect plants by root colonization and deplete all the resources from the rhizosphere that are available and accessible to RKNs' growth and development.Such an important relationship between microbes and plants found in the rhizosphere enhances plant health or helps the plant overcome abiotic or biotic stress [133].

Production of hydrolytic enzymes
Production of hydrolytic enzymes by rhizobacteria is also an important approach to controlling RKNs and other harmful pathogens effectively.Few Bacillus spp.produces various types of enzymes and enzyme complexes viz., chitinases, proteases, lipases, and collagenases.These enzymes affect various stages of PPNs and kill them.Chitinases enzyme produced by the bacterial species, B. pumilus, Serratia marcescens, and B. subtilis degrade cuticle and the outer surface of eggs and J2s of RKNs [134,135].Collagenases are another A. Khan et al. potent lytic enzyme produced by the B. cereus against the J2s of M. javanica [134].Some other groups of lytic enzymes like Glucanases, cellulases, and pectinases produced by Pseudomonas against M. incognita [134].

Induced systemic resistance (ISR)
ISR is one of the possible modes of action that induces resistance in host plants by activating the nematicidal compounds and preventing infection by nematodes.Several bacterial genera like B. pasteurii, B. subtilis, B. cereus, B. pumilus, B. amyloliquefaciens, P. putida, S. marcescens, P. fluorescence, and Rhizobium leguminosarum can provoke ISR [136].ISR against M. javanica and M. graminicola by fluorescent pseudomonads has been well documented [137,138].The antibiotic compound 2,4-diacetylphloroglucinol (DAPG) found as a systemic resistance-inducing agent in plants [139,140].However, VOCs and cyclic lipopeptides are also the key activators of ISR by endospore-forming gram-positive bacteria.

Bacterial species and their modes of action 8.2.1. Bacillus spp.
Several studies suggested that Bacillus spp.acts as an important bio-pesticide to control RKNs and improve plant growth.Antinemic action of Bacillus spp.based on their enzymes, toxic proteins, and antibiotic compounds they possess for example, antibiotics production by B. subtilis, and B. cereus, toxic proteins production namely Cry proteins by B. thuringiensis and enzymatic action by B. firmus [141].According to Li et al. [142], the application of B. cereus (strain BCM2) in tomato plants reduces the damage and population of M. incognita by 67.1 % compared to untreated plants.Few other strains viz., B. cereus (strain Jdm1) enhance the growth of tomato plants by inhibiting egg hatching, root galling (43 %), and the number of M. incognita J2s in vivo [143].B. pumilus (strain L1) is another example with anti-RKNs potential, produces chitinases and protease enzymes that degrade and digest the cuticle of M. arenaria [144].Culture supernatant and crude extract of B. amyloliquefaciens (strain Y1) were evaluated to control M. incognita by inhibiting egg hatching and increasing J2s mortality both in vivo and in vitro on tomato plants [145].
Bio-control of RKNs and other PPNs attributed to B. thuringiensis (Bt) that includes the formation of Cry protein or crystal protein (proteinaceous protoxin crystals).It has been found that several crystal proteins obtain from transgenic plants and their application to protect plants from RKNs [78].Presently 3 families of crystal proteins like Cry5, Cry6, and Cry55 cause the killing and growth retardation of nematode J2s [146].Cry5 is the most effective and potent crystal protein among all Cry proteins that causes the lysis of intestinal tissues of nematodes.

Pasteuria penetrans
Pasteuria penetrans is an endospore-forming bacterium, that parasitizes the J2s of RKNs [147].J2s with bacterial spores become immobile and unable to penetrate the roots [147].Bacterial spores attached to the nematode's cuticle, enter the body by germination tube, form endospores inside, and finally lead to nematode death (Fig. 5; Table 4).

Role of bacteria in plant growth promotion other than bio-control mechanisms
Plant growth-promoting bacteria (PGPB) includes a diverse group of microorganisms that represent a broad range of genera.Few genera viz., Pseudomonas, Bacillus, Lactobacillus, and actinobacteria are involved in plant growth promotion [156][157][158][159].They perform growth promotion, advancement, and development of plants by increasing beneficial microbiota in the vicinity of the rhizosphere, root colonization, competition with native harmful microbes, and build-up resistance in host plants against RKNs [160].Furthermore, Herbaspirillum, Azospirillum, Acetobacter, Serratia, Paenibacillus, Burkholderia, and Rhodococcus have also been found to increase crop growth and production [161].According to Chakraborty et al. [162], rhizobacterial strains viz., B. pumilis and B. amyloliquefaciens demonstrated in vitro plant growth-promoting attributes, like siderophores production, phosphate solubilization, Indole-3-acetic acid (IAA) production and antagonisms against RKNs.Such strains also favour an increment in the growth and yield of tea crops in terms of root and shoot biomass and the number of leaves in the field conditions [162].Siderophores production by plants and bacterial strains under low iron (Fe) conditions is also an important plant growth-promoting factor [163,164].Besides siderophores, IAA is also the most studied auxin formed by PGPR plays a key role in plant growth by involving in plant-microbe interactions, changes in the transcriptional hormones, cell wall-related and defence-related genes [165,166].It also induces root biomass, reduction in stomatal density and size to prevent water loss, and activates auxin response genes to improve plant growth [167,168].

Conclusions and future outlooks
Agriculture is an important part of the economy in many countries.Most of the human population depends on crops for their food.However, several crops (vegetables and fruits) are severely attacked by RKNs.When evaluating the significant damage caused by RKNs and the limitations made by the government for the use of pesticides, it is necessary to scientists and researchers to invent novel ecofriendly approaches to control RKNs.According to previous studies on nematode management, it has been found that the excessive use of chemicals polluted the environment, soil, and water, and also influences human health.Therefore, by considering these all problems, microbial bio-control emerged as a potent substitute for chemicals.
Several microbes such as fungi, and bacteria with their different strains have great nematicidal action.Fungi possess various modes of action against nematodes such as digestion and absorption of nematode cuticle and their penetration, rhizospheric colonization, and induction of resistance in plants.Whereas, bacteria counter-attack the RKNs parasitism through antibiosis (production of toxic compounds), rhizospheric competition with nematodes, production of several lytic enzymes (viz., collagenases, proteases, lipases, and chitinases), and induction of systemic resistance.Besides bio-control mechanisms, fungi, and bacteria also enhances plant growth and yield by increasing nutrient (N and P) uptake from the soil and nitrogen fixation as well.
Bio-control of RKNs has been around for the last two decades, but it is unable to achieve better results and more attention as new species are identified and examined as bio-control agents against RKNs.Currently, scientists are engaged in RNA sequencings involving

Table 4
Modes of action of bacterial species and their strains against root-knot nematodes on agricultural crops.16S and 18S rDNA sections, having great potential for the detection of new BCAs against RKNs.This approach will make bio-control studies faster, cheaper, more effective, and more practical in the future.Furthermore, it would be also helpful to focus on environmental health, soil fertility, and non-target microbial diversity during nematode management.

ROS Reactive oxygen species SMs
Secondary metabolites ISR Induced systemic resistance VOCs Volatile organic compounds

A
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Fig. 4 .
Fig. 4. Modes of action of Trichoderma spp. as bio-control agent against root-knot nematodes on agricultural crops.

Fig. 5 .
Fig. 5. Modes of action of bacteria as bio-control agents against root-knot nematodes on agricultural crops.

Table 1
Worldwide losses on agricultural crops due to root-knot nematodes.
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